Method and apparatus for flow cytometry

ABSTRACT

A flow cytometer ( 20 ) capable of utilizing an imaging system including a sensor ( 88 ) to determine various properties of the flow cytometer ( 20 ). Importantly, the imaging allows for determination of a drop delay time ( 154 ). Other characteristics that can be determined include at least the width of the stream, pressure of the stream, effect of charged droplets on other droplets ( 44 ), trajectory of the stream, resonant frequency, wavelengths, change in position of droplet break-off point ( 150 ), and others. An automatic warning system ( 180 ) can be used to alert an operator of an anomaly during setup or during normal operation. Furthermore, a mechanical interrupter can be used to shield a sort result from contamination by an incorrectly functioning stream. The cytometer ( 20 ) also allows for the disablement of the sort, particularly the charging and deflection systems. In addition, a more accurate system for detecting the speed of the stream can be used. The imaging system including a sensor ( 88 ) can allow for the removal of background noise and the monitoring of a change in a stream characteristic.

This application is the United States National Stage of InternationalApplication No. PCT/US99/04183, filed Feb. 26, 1999, which is acontinuation-in-part of U.S. patent application Ser. No. 09/032,733,filed Feb. 27, 1998, now U.S. Pat. No. 6,248,590, issued on Jun. 19,2001, each hereby incorporated by reference.

I. TECHNICAL FIELD

This invention relates to a method and apparatus for the analyzation andsorting of particles, e.g., in a flow cytometer. In the field of flowcytometry it is common to establish a stream of sheath fluid with astream of particles suspended in that sheath fluid. This stream can thenbe perturbed such that droplets form and the particles are contained inthe droplets as they break-off from the end of a contiguous stream. Thedroplets can then be sorted as desired by detecting desired particlesand establishing a charge on an individual droplet just before it breaksaway from the contiguous part of the stream. The droplet containing thedesired particle can then be deflected with an electric field into acollection container. As part of this process, it is optimal to knowwhen a droplet containing a desired particle reaches the charginglocation such that that particular droplet can be charged while dropletscharging as few neighboring droplets as necessary. This allows thedroplet containing the desired particle and possibly a few droplets oneither side of the droplet containing the particle—to be deflected intoa separate container and sorted out of the stream.

As part of this process it has been necessary to set up a flow cytometeron a daily basis and to allow that flow cytometer to equilibrate to theenvironmental conditions where the flow cytometer is located. This takesapproximately an hour to an hour and a half just for the process ofequilibrating the flow cytometer. Then, typically another one-half houris required to calibrate the drop delay timing of the flow cytometerafter the equilibration period expires. Therefore, a full one to twohours is required on a daily basis just for setup of the flow cytometer.This is time that could be used for producing results from the flowcytometer rather than wasting it on setup time. Therefore, there is adesire for a flow cytometer that does not require this one to two hoursetup time and that can be implemented quickly without the need forequilibration and calibration.

Another drawback to the present state of flow cytometry is the lack ofan automatic means of compensating for change in one of the parametersof the flow cytometer—most importantly, the drop delay time. Forexample, it is currently necessary for a technician to monitor a sortingflow cytometer during the process of sorting. The technician must remainin the room while the sort is being performed in case a catastrophicfailure of the flow cytometer would occur. In such a case, thetechnician could then, as quickly as possible, interrupt the sort andprevent any gathered sample of cells, for example, from beingcontaminated during a catastrophic failure. This might occur, forexample, if a nozzle becomes clogged and the stream is angled away fromthe nozzle tip toward one of the sample collectors. Even with atechnician in the room watching the sort take place, it would stillrequire possibly two to three seconds for the flow cytometer to bestopped. In the case of some types of sorts, however, even this two tothree second period would be too long to save the sort. Therefore, theprocess would have to be re-started and performed again. This can bequite frustrating—particularly if the sort had been near completion.

Furthermore, currently no warning system appears to exist when aparameter of the flow cytometer is set up in an incorrect manner. Forexample, if an incorrect nozzle size has been put on the flow cytometer,no manufacturer appears to be issuing a warning that can be used toalert the technician that the wrong nozzle size is attached. Therefore,this can result in unnecessary time on the part of the technician intrying to determine the problem with the setup of the flow cytometer.

Another drawback to the present state of the art in flow cytometry isthe inability to determine a drop delay time for a particle to thedegree of precision desired. Presently, one method that is used is toestablish the stream and strobe the stream with a light source such thatthe stream can then be viewed on a monitor to see if the dropletbreak-off point of the stream changes position. If the break-off pointshifts, then the stream can be re-calibrated to set the drop delay timefor a particle. This is deceptive however, because a change inwavelength of the stream might occur without a resulting change in thedroplet break-off point. Consequently, the drop delay time for aparticle would change—as the change in wavelength would indicate achange in speed of the fluid flow. However, this would go unnoticed by atechnician who was relying on the droplet break-off point position. Italso assumes that the hydrodynamics of the stream are constant once thestream leaves the nozzle of the flow cytometer and thus, assumes thevelocity of the stream remains constant.

II. BACKGROUND

Prior work in the field of flow cytometry apparently has beenunsuccessful in solving these problems. Furthermore, they have focusedon maintaining the droplet break off point—rather than appreciating theability to determine a drop delay time for a particle. For example, U.S.Pat. No. 4,691,829 to Robert E. Auer tried to utilize a laser beam aimedat the stream above the droplet break-off point. Based on refractiveproperties of the stream, it was then attempted to detect changes in thesurface of the stream. A change in the undulations of the surface couldthen be used to determine when the break-off point had shifted. However,this method did not actually determine a drop delay time for a particledetected in the stream. It merely tried to maintain the dropletbreak-off point at the same position. Furthermore, it required verysensitive equipment to detect the change in the undulation of thesurface and has apparently since the patent issued in 1987 never beenmade to work in a commercial product.

An earlier attempt to try to control the droplet break-off point can beseen in U.S. Pat. No. 3,761,941. In that patent, a test sample was runthrough the cytometer to try to detect a charge on a droplet. Atheoretical charge that was expected to have been applied to the dropletwas then compared to the actual charge on the droplet. The amplitude ofthe drop stimulating disturbance was then adjusted until the actualcharge approached the theoretical charge. In this manner, the streamcould be adjusted to the correct point for charging purposes.

In 1982, U.S. Pat. No. 4,361,400 discussed the use of a televisionmonitor to view the breakoff point of a cytometer. However, it alsorequired the operator to manually adjust the settings of the cytometerbased on the viewed breakoff point. Therefore, equilibration of thecytometer was still likely a one and a half hour procedure if thismethod were used.

In 1997, U.S. Pat. No. 5,700,692 discussed the use of a camera/monitorsystem to allow a user to adjust the distance between droplets in acytometer. However, it did not appreciate the ability of a monitoringsystem to determine a wealth of other characteristics of a stream andthereby automatically provide feedback to the flow cytometer. Instead,it focused on determining a center of mass of droplets and assumed aconstant velocity of the fluid stream. In focusing on the center of massof droplets, it apparently completely overlooked important informationthat could be determined from the stream—including an automaticregulation of a drop delay time.

Consequently, there is still a need for a flow cytometer that canmonitor a stream of the cytometer and detect a drop delay time based onthe specific characteristics of the stream at a specific point in time.Rather than relying on an expected steady state condition, such as aconstant velocity of the stream, there is a need for a cytometer thatcan determine the drop delay time under the specific conditions of thestream for a particular particle that is about to be sorted.Furthermore, there is a need for a flow cytometer that can adjust thedrop delay time at the beginning of the day when the flow cytometer isstill adjusting to environmental conditions such as room temperature. Inthis way, the flow cytometer can be used for useful sorts during thefirst one to two hours that were previously required for equilibrationto environmental conditions and calibration of the flow cytometer, suchas calibration of the drop delay time using a standard test sample.There is also still a need for a flow cytometer that can detect when acatastrophic event occurs that could result in the destruction of anearly completed sort—for example a five to six hour sort that iscontaminated when a nozzle becomes blocked and the stream isinadvertently diverted into the sample collection container. Inaddition, there is still a need for an automatic interrupter that candivert or block a stream or turn off the sorting aspect of the streamautomatically upon the occurrence of an event such as a catastrophicfailure. In this manner the collected sample could be protected in asfast a time as possible, especially faster than the two to three secondsthat would be required if an operator were to do it by hand—as isapparently the case with current cytometers.

In addition, there is a need to understand the characteristics of thespeed of the stream that is ejected by a flow cytometer—especially fromthe time that the stream is ejected from the flow cytometer through thepoint where a droplet is charged so that a charge can be applied at thedroplet when the droplet reaches the charging location. In the past, ithas been assumed that the speed was constant. However, as throughput isincreased and particles become closer to one another in the stream, itis even more critical to be able to determine the speed of the streamdrop delay time as accurately as possible; therefore, it is equallycritical to understand the characteristics of the stream rather thansimply estimate the stream as having a constant velocity.

III. DISCLOSURE OF INVENTION

The present invention provides a novel method of compensating forchanges in a flow cytometer and accurately determining a drop delay timefor a particular particle in the stream. A detected droplet can becharged based on a drop delay time that is computed based on ameasurement of the speed of the fluid in the stream. Therefore,knowledge of the droplet charging location and the speed characteristicsof the stream allows the cytometer to more accurately charge the dropletcontaining the particle at the droplet charging location.

In addition, other embodiments of the invention allow an image of thestream to be captured to determine information about the flow cytometer.For example, an image of the stream can be used to determine the widthof the stream at a given point and correlate this to a nozzle size beingused by the cytometer. In this fashion, it can be determined whether thecorrect nozzle size is being used. Other parameters can be determined inthis fashion as well. For example, a velocity of the stream can bedetermined at various points along the stream. A velocity of the streamcan be determined by measuring a wavelength of a surface wave on thestream and knowing the oscillation frequency that is being used by themechanical device perturbing the stream in order to calculate thevelocity of the stream at that point. Or, droplets occurring below thedroplet break-off point can be imaged and a distance between the twodroplets determined in order to determine the speed of the stream at thedroplet break-off point.

Furthermore, an exponentially decaying model can be used to model thechange in velocity of the stream below the nozzle exitpoint—particularly between the nozzle exit point and the dropletbreak-off point for the stream fluid. This model can then be used todetermine a more exact drop delay time for a particle detected in thestream.

In addition, the image monitoring system can be used to image thedroplet break-off point to determine a location of the droplet break-offpoint, as well as a change in position of the droplet break-off point.Furthermore, the system can be used to provide a feedback signal to theflow cytometer such that the droplet break-off point is re-establishedor maintained at the desired position.

Also, the image monitoring system can be used to determine the effect ofa charged droplet on a successive droplet or droplets. This can beaccomplished by monitoring the trajectory of a droplet in real time andcharging the subsequent droplet with a slight charge that allows it tofall in line with other droplets that do not contain particles.

The imaging system can also be used in a similar manner to determine themost stable position of the stream for a given pressure and oscillation.In this manner the preferred resonant frequency for a stream can beselected and the resulting stream established in a stable position thatwill not require oscillating between positions.

The invention can also be used to warn an operator of the flow cytometerthat an anomaly exists in the setup or operation of the flow cytometer.In this fashion, the cytometer can perform a self-test during setup aswell as during operation. For example, a determination of the speed ofthe stream could be used to determine whether the correct hydraulicpressure is being used for the flow cytometer. Or, a warning can beissued to the operator inquiring whether a different nozzle size isintended based on a width of the stream, for example. Similarly, a shiftin position of the stream could be used to determine if the nozzle hasbecome clogged or whether some other anomaly exists with the cytometer.Furthermore, if the stream is detected to have disappeared completely,the cytometer could warn the operator that a catastrophic event thatwould damage the sort had occurred. These warnings might be issued toeither a remote monitor, a paging device, alarm device, or even ane-mail message.

Furthermore, rather than simply issuing a warning to the operator,mechanical intervention can be utilized to automatically or manuallydivert the stream and prevent it from contaminating a gathered sample.This might be accomplished using either a gutter or deflector.Alternatively or concurrently, one embodiment of the invention allowsthe sort to be disabled, either manually or automatically, by disablingthe charging of the stream and/or disabling the deflection force thattypically deflects a charged particle.

The invention also utilizes imaging methods to remove electrical noisefrom the captured images. For example an image of the stream can becaptured and outlined to establish a first background image of thestream and then used as a template for comparison of subsequent imagesof the stream. In this fashion, electrical noise can be removed onsubsequent images and the outline of the images compared to see if therehas been a change in a characteristic of the stream.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram of an apparatus of one embodiment ofthe present invention;

FIG. 2 is an alternative diagram of a closeup view of a flow cytometerstream;

FIG. 3 is a view of a stream captured by a camera and displayed on amonitor with a pixel-based display;

FIG. 4 is a first alternative of a flow cytometer with a mechanicalinterrupter;

FIG. 5 is a second alternative of a mechanical interrupter for a flowcytometer.

V. BEST MODE FOR CARRYING OUT THE INVENTION

Referring now to FIG. 1, a preferred embodiment of the invention can beseen in detail. The flow cytometer (20) can utilize a source of streamfluid (24) to supply stream fluid to establish a sheath of fluid inwhich particles (32) can be suspended. The source of particles (28) caninsert the particles from time to time such that the particles becomesuspended in the stream fluid and are hydrodynamically focused in thestream. A stream (36) comprised of the stream fluid (40) and theparticles (32) can then be established below the nozzle (64) of the flowcytometer. The stream can be established in a steady state conditionsuch that droplets (44) are formed and break away from a contiguous partof the stream. When the stream is established in this steady statefashion, a stable break-off point (48) can be established. This streamcan be strobed with a stroboscope to illuminate the stable stream. Atthe break-off point the stream breaks off into droplets with thesedroplets centered about the break-off point. The droplet break off pointis the point in a stream where a droplet separates from the contiguousflow of the stream. For reference purposes, the center of the dropletwill be considered the droplet break off point when it is necessary todefine the point in such an exacting manner. Below the droplet break-offpoint (48) a free fall zone (52) can exist. This free fall zone embodiesthe area where the droplets move once they break away from thecontiguous part of the stream. At the bottom of the nozzle (64) a streamexit point (56) is established. The exit point is the point in spacewhere the stream emerges from the flow cytometer. For example, the exitpoint on a flow cytometer having a nozzle would exist where the streamexits from the nozzle. At this point the stream essentially emerges oris ejected from the flow cytometer. A droplet charging location (60) canexist at a point along the stream. This droplet charging location (60)can exist, for example, at the droplet break-off point (48) as seen inFIG. 2. As a possible alternative, a charging ring can be used andpositioned below the droplet break-off point such that the individualdroplets can be charged. An oscillator (68) as shown in FIG. 1, can beused to perturb the stream and to establish a steady state oscillationof the stream. It is preferred to use a piezoelectric crystal toaccomplish this perturbation of the stream. The oscillator (68) may havean adjustable oscillation frequency that can be adjusted to perturb thestream at different frequencies such that droplets are created atdifferent rates. Furthermore, it can be used in conjunction with thestream pressure to establish the rate of droplet formation.

A detector (72), such as a laser and receiver in combination, can beused as seen in FIG. 1 to monitor the stream for a particle. Thedetector can detect the particle in the stream as the particle passesthrough, for example, a coherent beam of light aimed at the stream bythe detector. When the coherent beam of light intercepts a particle inthe stream, fluorescence or scattered light rays can then be emitted ordeflected, respectively, as shown in FIG. 2 to a receiver of thedetector. Alternative methods of detection are also well understood bythose of ordinary skill in the art.

As the droplets fall in the free fall zone, they can pass through asorting force generator (80) such as electrostatic plates shown in FIG.1. If the droplets have been charged with a positive or negative charge,an electric field established between these electrostatic plates willdeflect the charged droplets such that the trajectory of the droplets ischanged. As seen in FIG. 1, these droplets can then be deflected into acontainer (76) which acts as a sample collector. Similarly, thosedroplets that are neutrally charged can fall into the center containershown in FIG. 1 and droplets that are alternatively charged can fallinto a third container. Furthermore, alternative techniques such asutilizing different quantities of charge can be used to accomplish aneven greater deflection.

To accomplish the charging of the droplets a charging device (84) can beused to charge the stream fluid. Therefore, when a particle is detectedand known to have reached the droplet break-off point, the stream can becharged such that when the droplet breaks away from the stream, itcontains a net charge. A net charge should be understood to mean eithera net positive, net negative, or even a neutral charge. Then, thecharging device can be turned off or even configured to produce anopposite charge such that subsequent droplets which have been inducedwith a charge by the droplet containing a particle are counteractivelycharged back to the steady state charge of the stream—typically aneutral charge.

A sensor (88) can be used to accomplish many of.the techniques of thepresent invention. Initially, the sensor (88) can measure a property ofthe flow cytometer based on a captured image of the fluid stream. Onesuch property that the sensor can measure is the speed of the stream ata point along the stream. Two points of particular interest are thespeed of the stream at the area just below the exit point of the streamfrom the nozzle and at about the droplet break-off position of thestream.

The sensor (88) can be oriented to measure a wavelength (92) of thestream as shown in FIG. 2. Digital imaging routines can operate on thewave shape to measure the length of a standing wave, for example.Furthermore, the sensor can be oriented to measure a distance betweendroplets of the stream such that the distance and the oscillationfrequency of the oscillator can be utilized to calculate the speed ofthe droplets at that point along the stream. The sensor (88) can utilizea camera (102) with a wide angle lens that captures a large portion ofthe stream or multiple cameras such as those seen in FIG. 2 where acamera (96) captures an image of the stream at an exit point of thestream and camera (98) captures an image at the droplet break-off pointof the stream. In this fashion, camera (96) and camera (98) can serve asa first sensor and second sensor, respectively, for determining speedsat different points along the stream. The camera (102) as shown in FIG.1, can capture an image (106) of the stream and display it on a monitor(114).

The camera (102) can be oriented to capture various features of thestream. For example, the camera can be oriented to capture the width(110) of the stream as shown in FIG. 3. Alternatively or additionally,the camera can be oriented to capture an image that permitsdetermination of a speed of the stream. As discussed earlier, this maybe accomplished by measuring the wavelength of the stream or measuring adistance between two droplets and utilizing the known oscillationfrequency to calculate a speed for the stream at those two points.Namely, the product of the oscillation frequency and the distancebetween droplets (or the stream wavelength) would yield a streamvelocity at that point.

In addition, the camera or sensor may be oriented to capture an imagethat permits determination of a change in stream position. This can bedone by monitoring a first position of the stream, recording thatposition, and then monitoring the stream over time to see if the streammoves away from that previously determined position. The camera cancapture an image of the stream at the break-off point as well as thestream exit point where the stream emerges from the cytometer.Essentially, the camera can be oriented in many different orientationsto capture many views in order to determine information about the streamwhich consequently allows the sensor to determine information about thecharacteristics of the cytometer. The various cameras can be used tocapture the image of the stream, however it is preferred to use a camerathat creates a digital representation for the image of the stream andone such camera being one that uses a charge coupled device or CCD. ACCD can produce an output in a series of analog voltage pulses each ofwhich corresponds to a light intensity received by a pixel of the CCD.

Once the sensor (88) captures an image of the stream, the image can bedisplayed on a monitor (114). The monitor can then display the image ofthe stream to a user. Typically, a monitor (114) will also be comprisedof individual pixel elements (118) that correlate to a digitalembodiment of the image derived from the CCD. The CCD's pixel elements,as well as the monitor's embodiment) can be correlated with a physicaldistance to arrive at an accurate determination of an actual dimensionof the stream. The monitor (114) can be positioned at the flowcytometer, or when it is desired to monitor the flow cytometer at aremote position, the monitor (114) can be positioned at such a remoteposition.

A memory device (122) can be used to store at least one parameter forthe flow cytometer. Once an image is captured by the camera (102), thememory device can also serve to store a representation of the stream,more particularly, a digital representation of the stream. An imagingmeans (126) can be used by the sensor for creating a digitalrepresentation of the stream. More particularly, a means for outlining(130) can be utilized to create an outline of the stream (134) as shownin the representation of the stream in FIG. 3. Similarly, this means foroutlining (130) can be used to create an outline of an individualdroplet (138). Digital representation is intended to mean arepresentation of the object. For purposes of this patent it will stillbe considered a representation when only a portion or an outline of theborder is used rather than the entire object. The means for outliningcan be comprised of a typical digital imaging processing program ordigital video processor which can operate on an image to detect actualsignal versus noise in the signal. Such digital imaging processingprograms can be seen by reference to: Digital Imaging Processing byKenneth R. Castleman, Prentice Hall Dec. 1, 1995; The Image ProcessingHandbook, by John C. Russ, CRC Pr. January, 1995; Digital ImageProcessing, by Rafael C. Gonzalez, Addison-Wesley Apr. 1, 1992; andAlgorithms for Image Processing, by James R. Parker, John Wiley & SonsNov. 1, 1996 which are hereby incorporated by reference. Furthermore,U.S. Pat. No. 5,700,692 discusses imaging techniques and is herebyincorporated by reference for the imaging techniques disclosed.

An analyzer (142) can be used to analyze an image of the stream in orderto determine information about the flow cytometer. For example, theanalyzer can determine a distance between objects. Furthermore, theanalyzer can determine a deflection of a charged droplet from thecentral axis of the stream based on the droplet position in the imageand the uncharged stream steady state position. This can be accomplishedby simply measuring the distance of the droplet from the steady statestream with a typical digital imaging processing program.

The analyzer (142) can also determine a charge on the droplet. Forexample, when one wants to know whether the proper charge was applied toa droplet the analyzer can be used to determine the actual charge ascompared to the anticipated charge on the droplet. This can beaccomplished by noting the deflection of the charged droplet from thestream noting the droplet size based on the image, measuring the mass ofthe droplet, which is a function of the droplet size and mass of theknown fluid, and the known electric field set up by the deflectionplates. In this fashion, the actual deflection can be used to deduce thecharge on the droplet and therefore that actual charge can then becompared to the anticipated charge to confirm whether the chargingprocedure is actually charging the droplets to the proper charge. Inaddition, a droplet sizes can be detected at the breakoff point and thecharging point adjusted to charge the droplets such that they will bedeflected the appropriate distance given their size.

The analyzer can also be used to calculate the best “defanning” chargebased on the deflection (or charge) of a previous droplet. For example,when one wants to establish a very steady stream of uncharged droplets,i.e., avoid a stream of uncharged droplets that make the stream appearlike it is fanning back and forth, it is often necessary to apply apartial charge to droplets neighboring charged droplets. This is due tothe fact that the charged droplets will induce a charge on neighboringdroplets. Therefore, by slightly charging droplets that occur after acharged droplet, the induced charge on the successive droplets caused bythe charged droplets can be counteracted. For example, if a positivelycharged droplet is expected to induce a negative charge on a successivedroplet, the successive droplet can be charged slightly positive tocounteract the induced effect.

The analyzer can also be used to measure the width of a stream whichallows a determination of nozzle size. For example, the stream width of60 microns (or 25 pixels on a 480×512 pixel image) can be determined toindicate a nozzle size of 70 microns. Furthermore, the analyzer can beused to determine the best resonant frequency for the cytometer. Forexample, the image can be analyzed to see where the stream establishesthe shortest break-off point and the oscillation frequency of theoscillator can be set to correspond to that point. Typically, this willbe a function of nozzle size and velocity.

Furthermore, the analyzer can be used to determine the speed of thestream based on distances determined in the image and the knownfrequency of the oscillator. To accomplish this analyzation process, theanalyzer (142) can utilize a computer (146). The computer (146) canutilize the approximate speed of the stream at a first position and theapproximate speed of the stream at a second position along the stream inorder to determine a stream characteristic by modeling the speed of thestream in a region occurring between the stream exit point and thedroplet break-off point as decaying approximately exponentially relativeto distance.

Furthermore, the analyzer can utilize a means for determining a changein droplet break-off position (150) to determine when the break-offposition of the stream changes.

This can be accomplished by recording a droplet break-off position andcomparing that position to subsequent break-off positions noted insubsequent images. A simple computer program can be used to accomplishthe means for determining a change in droplet breakoff position, bystoring a representation of the droplet breakoff position, capturing asecond digital representation and then comparing the two representationsto see if they correspond.

The analyzer can also use a means for determining drop delay (154), thedrop delay being associated with a detected particle. A drop delay (ordrop delay time) is considered to be the delay in time between detectinga particle in the stream and acting upon the droplet in which theparticle is contained in order to accomplish a sort of the particle. Forexample, in a typical flow cytometer, a particle will be detected by adetector and characteristics of the particle will be determined based ona fluorescence of light from the particle. Then, based on thecharacteristic, the droplet the particle is in will be charged justbefore it breaks away from the stream and sorted by an electrostaticsorter. The drop delay in this instance can be considered to be the timebetween detection of the particle and charging of the droplet. Inanother embodiment, one might choose to charge all the droplets, butonly apply the electrostatic field for the particle/droplet to besorted. In this case, the drop delay would be the time between thedetection of the particle and the act of applying the electrostaticfield. Similarly, the drop delay might be initiated at a point in timeafter the first detection of the particle, such as the time when theparticle fluoresces. In this case, the drop delay might be calculated asthe time between detection of a florescence and the time for theparticle to reach the charging location. Similarly, for embodiments thatuse a charging ring to charge a droplet separate from the stream, thedrop delay might be calculated from the point of particle detection tothe point where charging occurs. Determination of the drop delay timecan be accomplished with a simple software program for instance bydetermining a speed of the fluid stream at a particular position andmodeling the speed of the stream based on an exponentially decayingspeed with respect to distance. Therefore, the software routine couldaccount for the change in speed of the stream over a distance and derivea time for the particle to traverse that distance to arrive at thecharging location. For example, a comparator (158) can be used tocompare determined information about the stream, for example, a measuredproperty of the flow cytometer to a parameter of the flow cytometer. Forpurposes of this application, a parameter is a pre-determined orexpected value for a characteristic of a thing, such as an expectedwidth of a stream in a flow cytometer, an expected nozzle size, anexpected break off position, an expected stream location, etc. On theother hand, and again for purposes of this application, a “property” isa quality, trait or quantitative value representative of a thing, suchas the measured width of the flow cytometer stream, the measuredpressure of the stream, the measured distance from a nozzle tip to thedroplet break off point, the measured temperature of the stream, theactual mass of the fluid used for the stream, etc. The means fordetermining drop delay can utilize a compensator (162) to compensate forchange in speed of the stream after the stream emerges at the exit pointof the flow cytometer. Essentially this compensator can be comprised ofa software routine that models the speed of the fluid as exponentiallydecaying, as those who are skilled in the art would easily understand.

A time delay generator (164) can be utilized by the flow cytometer (20)to provide a delay in charging the droplet which contains the detectedparticle once the particle is detected in the stream. Based on theresults from an analyzer, the time delay generator can be set and usedto control the droplet charger (84). A signal generator (168) can beused by the analyzer to generate a signal to the flow cytometer or toexternal indicators based on the determined information from the stream.For example, the signal generator can generate a signal used tore-establish the droplet break-off position. Furthermore, the signalgenerator can issue an error warning about the flow cytometer based onthe determined information from the stream. In addition, the signalgenerator can be used to generate a signal to the flow cytometer basedon the comparison of the first digital representation of the stream anda second digital representation of the stream as explained above.

FIG. 1 shows a remote paging device (172) that can be located with anoperator of the flow cytometer when the operator leaves the area wherethe flow cytometer is located. Given the advancements of the presentinvention which allow the flow cytometer to detect catastrophic events,it is possible for an operator to leave the flow cytometer unattendedand to perform activities elsewhere. The remote paging device can beused to warn the operator, for example, that a catastrophic event, i.e.,an event which requires a cessation of sorting such as total loss of thestream, clogged tubes or nozzle, or air in the cytometer chamber, hasoccurred so that the operator can return to the flow cytometer andattend to whatever problem may exist. A remote computer monitor (176)can also be utilized with the flow cytometer to provide a similarwarning. For example, the flow cytometer could be connected on a WindowsNT platform such that a pop-up message can be displayed on an operator'sterminal indicating the completion of a sort or a problem with the flowcytometer. Similarly, the flow cytometer can be connected to an E-mailsystem such that an E-mail message can be automatically routed to a useror interested party.

Alarm circuitry (180) can be utilized to indicate an alarm condition.Such circuitry may be comprised of an alarm (184) which can assume avariety of configurations such as a displayed message, a flashing light,a buzzer, or other commonly used devices.

One novel feature of a present embodiment of the invention is the use ofa mechanical interrupter (188) which can be utilized to intercept and/orinterrupt the stream. For example, the mechanical interrupter (188) maybe utilized after the stream exits from the exit point of the flowcytometer such that the stream can be interrupted and diverted away ordeflected away from its normal course or from the abnormal course thatis caused due to a problem with the flow cytometer. The mechanicalinterrupter can be used to interrupt the stream based on a determinationmade by the sensor. In this fashion it can work automatically. Onepossible embodiment of the mechanical interrupter can utilize a gutter(192) as shown in FIG. 4 which routes the stream away to a wastecontainer after the gutter swings in to intercept the stream.Alternatively, a deflector (196) can be used to swing into position suchthat the stream is deflected away to the waste container. Preferablythese mechanical interrupters are located close to the exit point of thestream such that the stream can be diverted as early as possible toavoid contamination of the collected samples. Both may swing or slide ineither manually or automatically.

With a background understanding of the apparatus of the presentinvention, the method of utilizing the apparatus to accomplish variousembodiments of the invention can now be better understood. Inparticular, one aspect of the present invention involves utilizing animaging system to capture images of the stream. Particularly the dropletbreak-off point of the stream can be captured as an image such that aspeed of the stream can be determined. This can be accomplished byimaging the stream and identifying droplets that form below the dropletbreak-off point and determining a distance using imaging techniquesbetween subsequent droplets. These droplets will be traveling at a speedvery close to the droplet that is located at the droplet break-offpoint. The image of the stream can be stored as a digital image inmemory, e.g., RAM, of the cytometer.

Imaging techniques can be utilized to analyze the stream. For example,electrical noise can be eliminated from a captured image. This mayinvolve capturing a first image of the stream at a location along thestream and creating a first digital representation of the stream basedon the first image. For example, an outline of the stream might becreated. Then that digital representation of the stream can be stored inmemory. For example, the outline can be stored in memory. Additionally,an outline of a droplet from the stream can be created and stored inmemory as well. Next, a second image of the stream at the same locationcan be captured and a second digital representation created. The seconddigital representation of the stream can be based on the second capturedimage. Then, the two digital representations of the stream can becompared and a determination can be made whether a property of thestream has changed based on the comparison of the first digitalrepresentation with the second digital representation. Standard digitalsignal processing techniques can be used to accomplish this as would beunderstood by those of ordinary skill in the arts.

Furthermore, one embodiment of the invention can determine informationabout the flow cytometer based on a captured image. For example, thismight be accomplished by measuring a property of the flow cytometerbased on the captured image. Such properties might include the width ofthe stream, the speed of the stream, the pressure of the fluid, thewavelength of a wave on the stream indicative of the stream speed, orother characteristics. Furthermore, information can be determined aboutthe flow cytometer by associating a physical distance with a digitalimage block or a pixel dimension. An actual physical distance can bedetermined based on the number of blocks or pixels used to representthat distance on the captured image.

The captured image can also be used to determine a velocity of thestream. This can be accomplished by measuring a wavelength of the streamat some position along the stream. Furthermore, the oscillationfrequency of the oscillator can be used in conjunction with the measuredwavelength to calculate an approximate speed of the stream. A velocityof the stream can be determined at an approximate exit point where thestream emerges from the flow cytometer by measuring a wave length thereand utilizing the known oscillator frequency. In addition, anapproximate velocity of the stream can be measured at about the dropletbreak-off point by imaging droplets and measuring the distance betweensuccessive droplets. By utilizing the oscillation frequency to determinethe time period between successive droplets and given the measureddistance an approximate velocity can be calculated for the dropletbreak-off point.

The captured image can also be utilized to determine a drop delay timefor a particle in the stream. This can be accomplished by determining anapproximate first speed of the stream at a location along the stream andthen determining an approximate second speed of the stream at adifferent point along the stream. Preferably a speed close to theparticle detection point would be utilized for the first speed of thestream and a measurement of the stream speed at approximately thedroplet break-off point would be utilized for the second speed of thestream. These two speeds can then be used as part of a model forpurposes of modeling the stream speed. Experimental results indicatethat the speed of the stream will approximately exponentially decay fromthe point where it is ejected from the flow cytometer to a typicaldroplet break-off point. However, an appropriate function of the speedcould easily be determined for a stream by generating calibration datafor stream once the stream was established and deriving a real functionfor the change in speed of that stream for the actual conditions. Atypical velocity of the stream at the exit point will typically, in oneembodiment of the invention, approach 27 meters per second, while at thebreak-off point the velocity will have dropped to 25 meters per second.This is believed to be due to hydrodynamic relaxation of the stream overthe distance from the exit point to the droplet break-off point. Inprevious attempts by others to determine an appropriate drop delay timethose earlier attempts had utilized a constant velocity throughout thisrange of the stream. Therefore, this probably resulted in a lessaccurate drop delay time and consequently a poorer result in charging adesired droplet. Consequently, once the first speed of the stream andthe second speed of the stream are measured, the stream speed can bemodeled as decaying approximately exponentially—as experimental datasuggest—in the region between the exit point and the droplet break-offpoint. Then, given the point where a particle is detected and a knowndistance to a charging point for the droplet containing the particle (oreven by determining that distance through imaging of the stream bydetermining the present droplet break-off point when the particle wasdetected or monitoring a shift in that droplet break-off point as theparticle descends in the stream), the flow cytometer can compensate forthe change in speed due to the exponential decay of the speed of thestream in the region. Then based on this modeling of the stream speedand the compensation for the change in speed the flow cytometer candetermine a time for the particle to flow over the distance to thecharging point. In this fashion, the approximate speed of the stream atthe first position along the stream and the approximate speed of thestream at the second position along the stream can be used to determinethe stream characteristic, namely in this example, a drop delay time forthe stream.

Once a drop delay time has been calculated that drop delay time can beutilized to calculate when the particle will reach a droplet charginglocation, e.g., based on the known point in time when the particle wasdetected. Then, the flow cytometer can charge the droplet containing theparticle as it reaches the charging location, for example, by chargingthe stream or by using a charging ring.

As noted earlier, a present embodiment of the invention can be used tomeasure properties about the flow cytometer in order the generatewarnings about the flow cytometer. This can involve defining at leastone parameter for the flow cytometer and comparing that parameter to ameasured property or characteristic of the flow cytometer. In view ofthis comparison the flow cytometer can then determine when the operationof the flow cytometer does not satisfy at least one parameter definedfor the flow cytometer.

For example, a warning can be generated after determining that a changein position of the droplet break-off point has occurred. In previoussetups, others have relied on a technician to constantly watch an imageof the stream to see if the droplet break-off point shifts in position.With the present imaging techniques that allow comparison of previousdroplet break-off points with successive droplet break-off points, thisprocedure can be automated such that a warning signal is generated to anoperator of the flow cytometer.

As another example, the flow cytometer allows for adjusting a drop delaytime so as to properly charge a droplet at the droplet break-off point.This can involve detecting the speed of the stream and the current knowndroplet break-off point at the point in time when the particle isdetected, and then calculating the drop delay time for that particularparticle or even adjusting that drop delay time again as a dropletbreak-off point change is sensed after particle detection.

Furthermore, a present embodiment of the invention is to constantlymonitor and re-establish the position of the droplet break-off point (orfiducial break-off point) (48) from the stream. The instant inventionallows the determination of the position of the droplet break-off pointto be ascertained relative to the fixed point of detection of theparticle in the stream by the particle detection system or detector, orlaser and receiver (72). This assessment of the position may beaccomplished by taking sequential high resolution scans or images with acamera or monitor (102) synchronized with the strobe phase and thedroplet charging phase (84) so as to record an image of sequentialdroplets as they reach the break-off point from the stream. The imagesof the droplets at the location of break-off is digitized(126) and heldin a memory device (122). Digitized data relating to the location of thedroplet break-off points is then compared electronically. The distancebetween droplet break-off points may be determined by the digital imageprocessing program. Then, as the droplet break-off point begins to shiftin either direction, a feedback signal may be generated to eitherincrease or decrease the amplitude of the oscillations from theoscillator (68) in order to re-establish the droplet break-off point inits previous position. The position of the droplet break-off point maybe re-established in this manner because the distance at which dropletbreak-off occurs from a stream is a function of the stream velocity(Vb), the disturbance amplitude, and a time constant which is a functionof surface tension, fluid density, jet diameter (d_(j))_(j) andwavelength. The disturbance amplitude (Va) is proportional to theoscillator voltage to the oscillator (68) through a coupling constant(Cc). Therefore, droplet break-off distance (Xb) which varies relativeto a fixed position due to fluctuation in stream velocity, may be heldconstant by varying the disturbance amplitude while wavelength, fluiddensity, surface tension and jet diameter are held constant. Theserelationships are summed up in the following equation:

Xb=Vb.Te(Vb,d)−Vb.log(Cc.Va)/γ

where Te is a delay correction due to the non-uniform velocity profileof the stream where

Te=k/Vb=332.0/Vb, and

γ is defined by a Rayleigh analysis where${\gamma = \sqrt{\left\{ \left( {{8 \cdot {\sigma/\left( {\rho \cdot d_{j}^{3}} \right)}}\left( {{\xi \cdot \left( {1 - \xi} \right)}{{I_{1}(\xi)}/{I_{0}(\xi)}}} \right\}} \right. \right.}},$

and where

ξ=π.d_(j).f/Vb, as such using standard Rayleigh parameters.

These functional relationships are used in conjunction with computersoftware and computer hardware to create a oscillation control devicewhich generates a signal which adjusts the voltage to the oscillatorwhich varies the oscillations to the stream in response to the variationin location of the fiducial break-off point. The variation ofoscillation to the stream thereby maintains the location of the fiducialbreak-off point. Failure to maintain the fiducial break-off point maylead to a computer controlled automatic suspension of sorting activity.

In another embodiment of the invention an alarm or warning based on theinformation in or determined from the captured image can be generated.Such an alarm or warning can be based on a comparison of the determinedinformation about the flow cytometer with an expected characteristic ofthe flow cytometer. This might involve issuing an error warning aboutthe flow cytometer based on a comparison of the determined informationand the expected characteristic.

The types of warnings that can be issued by the flow cytometer can beseen through the following examples. For example, it might be desirableto compare an expected stream width, such as stream width (110) shown inFIG. 3 with a predetermined stream width expected for use in aparticular set-up or for a particular particle to be sorted. In thisfashion if an expected stream width is expected and a different streamwidth is measured, a warning can be generated to the user or operatorindicating that an incorrect nozzle size may be connected to the flowcytometer. Similarly, the error warning might indicate that an improperpressure is being used. In addition, as noted earlier, the imagingtechnique can be utilized to determine a speed of the stream and thisdetermination of the stream speed might be utilized to issue a warningabout the pressure of the stream. If an expected stream pressure wouldcreate a predetermined stream speed and a measured stream speed weredifferent from that predetermined stream speed, then an error warningmight be appropriate. One significant aspect of the present invention isits ability to react to a catastrophic failure of the cytometer. Thismight be most noticeable through a significant change in position of thestream due to clogging of the nozzle. The imaging of the stream wouldallow a steady state position of the stream to be recorded and comparedto subsequent images of the stream. When a clogged nozzle, for example,diverts the stream at an angle, the imaging technique of comparingsubsequent images to a steady state image would allow the flow cytometerto determine that a catastrophic event was occurring and issue a warningto the user or even automatically interrupt the sorting process in orderto perfect the previously sorted sample. The warning can be generated ina variety of ways. One such way might be through displaying a warning ona remote computer monitor where an operator is working. Furthermore, awarning might be issued to a paging device.

Another significant embodiment of the invention allows the flowcytometer to disable the source based on information determined from acaptured image. For example, given the catastrophic failure detectedearlier due to a diversion of the stream or even the complete loss ofstream, the flow cytometer can act by mechanically interrupting thesort. This can be accomplished by automatically moving, swinging, orsliding a gutter as shown in FIG. 4 so as to intercept the stream andthen channeling away the stream in the gutter to a waste receptacle.Alternatively, this mechanical interruption might be caused byautomatically moving, sliding, or swinging a deflector, for example, aplate, in order to deflect the stream away from the collected samplethat was already produced by the flow cytometer. Alternatively, orperhaps even in addition to, where the stream is still present, the flowcytometer can automatically act to turn off the charging device or thedeflection source such that the charged particles are not deflected intothe collected sample.

Finally, one embodiment of the invention can be utilized toautomatically issue an alarm signal. In this fashion an operator can benotified that the flow cytometer has an alarm condition, for example,any abnormal condition. Such alarm signals might utilize either anaudible or visual alarm.

The foregoing discussion and the claims that follow describe thepreferred embodiments of the present invention. Particularly withrespect to the claims it should be understood that changes may be madewithout departing from the essence of the invention. In this regard, itis intended that such changes would still fall within the scope of thepresent invention. It is simply not practical to describe and claim allpossible revisions which may be accomplished. To the extent suchrevisions utilize the essence of the present invention, each naturallyfalls within the breadth of protection encompassed by this patent.Further, it should be understood that various permutations andcombinations of the elements shown in the claims are possible and shouldfall within the scope of this disclosure. In addition, it should beunderstood that the use of the word “comprising” is intended to have aninclusive meaning rather than an exclusive meaning. Therefore, inforeign countries, such as Australia, where this application may berelied upon as a priority document, the meaning of the word “comprising”is intended to have an inclusive meaning.

The discussion included in this application is intended to serve as abasic description. The reader should be aware that the specificdiscussion may not explicitly describe all embodiments possible; manyalternatives are implicit. The market place and manufacturing concernsmay dictate the appropriate embodiments for the present invention.Particularly with respect to the discussion, it should be understoodthat a number of changes may be made without departing from the essenceof the present invention. In this regard, it is intended that suchchanges—to the extent that they substantially achieve the same resultsin substantially the same way—will still fall within the scope of thepresent invention. It also may not fully explain the generic nature ofthe invention and may not explicitly show how each feature or elementcan actually be representative of a broader function or of a greatvariety of alternative or equivalent elements. Again, these areimplicitly included in this disclosure. Where the invention is describedin apparatus-oriented terminology, each element of the apparatusimplicitly performs a function. Apparatus discussions or claims may notonly be included for the systems described, but also method or processclaims may be included to address the functions the invention and eachelement performs. As but one example of this aspect, the disclosure of a“sensor” should be understood to encompass disclosure of the act of“sensing”—whether explicitly discussed or not—and, conversely, werethere only disclosure of the act of “sensing”, such a disclosure shouldbe understood to encompass disclosure of a “sensor.” Although themethods related to the system are being included in various detail, onlyan initial discussion directed toward the biosensor has been included.Naturally, that discussion could have some application to the variousother methods and aspects discussed throughout the disclosure. Neitherthe description nor the terminology is intended to limit the scope ofthe claims which will be included in a full patent application.

What is claimed is:
 1. A method of flow cytometry comprising:establishing a stream which emerges from a nozzle at a stream exitpoint, wherein the stream has a droplet break off point at whichdroplets break off and a free fall zone in which the droplets fall;determining an approximate speed of the stream at a first position alongthe stream; determining an approximate speed of the stream at a secondposition along the stream; and modeling the speed of the stream asdecaying approximately exponentially relative to distance in a regionoccurring between about the stream exit point and about the dropletbreak off point.
 2. The method of flow cytometry as described in claim 1further comprising the step of utilizing the approximate speed of thestream at the first position along the stream and the approximate speedof the stream at the second position along the stream to determine astream characteristic.
 3. The method of flow cytometry as described inclaim 1 further comprising the steps of: measuring a wavelength of thestream; utilizing an oscillation frequency of an oscillator of the flowcytometer; and calculating an approximate speed of the stream at ameasured wavelength position based on the measure of the wavelength andthe oscillation frequency.
 4. The method of flow cytometry as describedin claim 3 wherein the step of measuring a wavelength of the streamcomprises measuring a wavelength of the stream at about the exit pointof the flow cytometer.
 5. The method of flow cytometry as described inclaim 1 or 4 further comprising the steps of: measuring a distancebetween droplets of the stream; utilizing an oscillation frequency of anoscillator of the flow cytometer; and calculating an approximate speedof the stream based on the measured distance between droplets and theoscillation frequency.
 6. The method of flow cytometry as described inclaim 3 wherein the step of measuring a wavelength of the streamcomprises measuring a wavelength of the stream at about the dropletbreak off point.
 7. The method of flow cytometry as described in claim 1or 4 further comprising the steps of: detecting a particle in thestream; determining a point in time when the droplet in which theparticle is located will break off from the stream; and charging thestream so that the droplet containing the particle is charged.
 8. A flowcytometer comprising: a source of stream fluid to establish a stream; astream exit point where the stream is emerges from the flow cytometer; adroplet break off point where the stream breaks off into droplets; afirst sensor to determine an approximate speed of the stream at a firstposition along the stream; a second sensor to determine an approximatespeed of the stream at a second position along the stream; and acomputer to utilize the approximate speed of the stream at a firstposition along the stream and the approximate speed of the stream at asecond position along the stream in order to determine a streamcharacteristic by modeling the speed of the stream in a region occurringbetween the stream exit point and the droplet break off point asdecaying approximately exponentially relative to distance.
 9. The flowcytometer as described in claim 8 further comprising: a sensor orientedto measure a wavelength of the stream; and an oscillator having anoscillation frequency to perturb the stream to generate droplets. 10.The flow cytometer as described in claim 8 further comprising a sensororiented to measure a wavelength of the stream at about the stream exitpoint of the flow cytometer.
 11. The flow cytometer as described inclaim 8 or 10 further comprising a sensor oriented to determine adistance between droplets below the droplet break off point.
 12. Theflow cytometer as described in claim 8 or 10 further comprising a sensororiented to measure a wavelength of the stream at about the dropletbreak off point.
 13. A method of flow cytometry comprising: establishinga stream comprising a flow of particles, the stream having a dropletbreak off point; creating a first droplet, the first droplet having anelectrical charge; capturing an image of the stream; creating a seconddroplet in proximity to the first droplet; determining an effect thatthe electrical charge on the first droplet will have on the seconddroplet; generating a signal for the flow cytometer based on thedetermined effect; and charging the second droplet to counteract thedetermined effect that the charge on the first droplet will have on thesecond droplet.
 14. A method of controlling a flow cytometer, comprisingthe steps of: establishing a stream comprising a flow of particles, thestream having a droplet break off point; capturing an digital image ofthe stream to determine a drop delay time for a particle in the stream;and generating a signal for the flow cytometer, wherein a time ofgeneration of the signal is adjusted based on the determined drop delaytime from the captured image.
 15. The method of controlling a flowcytometer as described in claim 14 and further comprising the step ofusing a CCD camera to capture the image.
 16. The method of controlling aflow cytometer as described in claim 14 and further comprising the stepsof: associating a physical distance with a pixel dimension; anddetermining a physical distance based on the number of pixels used torepresent that distance in the captured image.
 17. The method ofcontrolling a flow cytometer as described in claim 14 further comprisingthe step of determining a speed of the stream at about an exit point,wherein the exit point is located where the stream emerges from the flowcytometer.
 18. The method of controlling a flow cytometer as describedin claim 14 or 17 further comprising the step of determining a speed ofthe stream at about the droplet break off point.
 19. The method ofcontrolling a flow cytometer as described in claim 14 further comprisingthe steps of: determining a speed of the stream at about an exit point,wherein the exit point is located where the stream emerges from the flowcytometer; and determining a speed of the stream at about the dropletbreak off point.
 20. The method of controlling a flow cytometer asdescribed in claim 14 further comprising the steps of: detecting aparticle; utilizing the drop delay time to calculate when the particlein a droplet will reach a droplet charging location; and charging thedroplet containing the particle at the charging location.
 21. The methodof controlling a flow cytometer as described in claim 14 or 20 furthercomprising the steps of: determining a first speed of the stream;determining a second speed of the stream; and utilizing the first speedof the stream and the second speed of the stream to model a change inspeed of the stream in order to determine the drop delay time.
 22. Themethod of controlling a flow cytometer as described in claim 14 furthercomprising the step of determining a change in position of the dropletbreak off point.
 23. The method of controlling a flow cytometer asdescribed in claim 22 further comprising the step of compensating forthe change in the droplet break off point so as to sort a droplet in adesired fashion.
 24. The method of controlling flow cytometer asdescribed in claim 22 further comprising the steps of: generating asignal to adjust the flow cytometer; and reestablishing the dropletbreak off point based on the generated signal.
 25. The method ofcontrolling a flow cytometer as described in claim 14 further comprisingthe step of eliminating electrical noise from the captured image.
 26. Aflow cytometer comprising: a source of stream fluid; a source ofparticles for suspension in the stream fluid; a stream comprising thestream fluid and the particles; a nozzle through which the stream isestablished; a break off point of the stream at which point the streambreaks off into droplets, said droplets centered about the break offpoint; a camera that captures an image of the stream; an analyzer thatanalyzes the image of the stream to determine the effect that a firstcharged droplet has on a second droplet; and a signal generator togenerate a signal based on the determined effect that the first chargeddroplet has on the second droplet.
 27. A flow cytometer comprising: asource of stream fluid; a source of particles for suspension in thestream fluid; a stream comprising the stream fluid and the particles; anozzle through which the stream is established; an exit point at whichsaid stream emerges from said nozzle; a break off point of the stream atwhich point the stream breaks off into droplets, said droplets centeredabout the break off point; a camera that captures a digital image of thestream; an analyzer that analyzes the digital image of the stream todetermine a drop delay time for a particle in the stream; and a signalgenerator to generate a signal, for the flow cytometer wherein a time ofgeneration of the signal is adjusted based on the determined drop delaytime.
 28. The flow cytometer apparatus as described in claim 27 whereinthe camera comprises a Charge Coupled Device.
 29. The flow cytometerapparatus as described in claim 27 and further comprising a monitor todisplay the stream, wherein the monitor is comprised of pixels.
 30. Theflow cytometer as described in claim 27 further comprising a sensor tomeasure a speed of the stream at about the exit point of the stream. 31.The flow cytometer as described in claim 27 or 30 further comprising asensor to measure a speed of the stream at about the droplet break offpoint.
 32. The flow cytometer as described in claim 27 furthercomprising: a sensor to measure a speed of the stream at about the exitpoint of the stream; and a sensor to measure a speed of the stream atabout the droplet break off point.
 33. The flow cytometer as describedin claim 27 further comprising a time delay to be used in delaying adroplet charger after a particle is detected in the stream.
 34. The flowcytometer as described in claim 33 further comprising a compensator tocompensate for a change in speed of the stream of the flow cytometerafter the stream emerges at the exit point of the flow cytometer. 35.The flow cytometer as described in claim 27 further comprising a meansfor determining a change in the droplet break off position.
 36. The flowcytometer as described in claim 35 further comprising a compensator tocompensate for a change in speed of the stream of the flow cytometerafter the stream emerges at the exit point of the flow cytometer. 37.The flow cytometer as described in claim 35 further comprising a signalgenerator to generate a signal used to reestablish the droplet break offposition.
 38. The flow cytometer as described in claim 27 furthercomprising an alarm to indicate an alarm condition.
 39. The flowcytometer as described in claim 27 further comprising a mechanicalinterrupter to intercept the stream after the stream exits at the exitpoint of the flow cytometer.
 40. A method comprising: establishing astream in a flow cytometer; capturing a first image of the streamestablished in the flow cytometer; creating a first digitalrepresentation of the stream based on the first image; capturing asecond image of the stream established in the flow cytometer; creating asecond digital representation of the stream based on the second image;comparing the second digital representation of the stream to the firstdigital representation of the stream; and determining whether a propertyof the stream has changed based on the comparison of the first digitalrepresentation of the stream with the second digital representation ofthe stream.
 41. The method as described in claim 40 further comprisingthe step of creating an outline of the stream.
 42. The method asdescribed in claim 41 wherein the act of creating an outline of thestream further comprises creating an outline of a droplet.
 43. Themethod as described in claim 41 or 22 further comprising the step ofstoring an outline of the stream in memory.
 44. The method as describedin claim 40, 41, or 42 and further comprising the steps of: comparingthe second digital representation to the first digital representation;and detecting a change in the representation of the stream between thefirst digital representation and the second digital representation. 45.A flow cytometer apparatus comprising: a stream source used to establisha stream; a camera to capture images of the stream; an imaging means forcreating digital representations of the stream; a memory to store afirst digital representation of the stream; a comparator to compare thefirst digital representation of the stream with a second digitalrepresentation of the stream; and a signal generator to generate asignal to the flow cytometer based on the comparison of the firstdigital representation of the stream with the second digitalrepresentation of the stream.
 46. The flow cytometer as described inclaim 45 further comprising a means for outlining to create an outlineof the stream.
 47. The flow cytometer as described in claim 46 whereinthe outlining means is configured to create an outline of a droplet.